# US 2020/0399636 A1 · Method, system and device for automated NGS library preparation

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- Number: US 2020/0399636 A1
- Document ID: US20200399636A1
- Status: Published application
- Kind: A1
- Application number: 16/907,237
- Filed: 2020-06-20
- Published: 2020-12-24
- Earliest claimed: 2019-06-20
- Inventors: Yan Wang; Lili Chen
- Web page: https://patentgrep.com/patent/US20200399636A1

## CPC

- C12N 15/1093: {General methods of preparing gene libraries, not provided for in other subgroups}
- C12N 15/1013: {by using magnetic beads}
- C12Q 1/6806: Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay (C12Q1/6804 takes precedence)
- B01L 2200/0642: Filling fluids into wells by specific techniques
- B01L 2200/0647: Handling flowable solids, e.g. microscopic beads, cells, particles
- B01L 2200/0668: Trapping microscopic beads
- B01L 2200/10: Integrating sample preparation and analysis in single entity, e.g. lab-on-a-chip concept
- B01L 2200/16: Reagents, handling or storing thereof
- B01L 2300/087: Multiple sequential chambers
- B01L 2400/0487: fluid pressure, pneumatics
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- C12Q: MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS (immunoassay G01N33/53); COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- B01L: CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE

## Abstract

Disclosed is an enclosed magnetic beads-based reaction system for automation of multi-step DNA preparation process involving DNA purification, modification and amplification. It is especially suitable for making DNA library preparation for next-generation sequencing. It uses modules to perform steps of the multi-step reaction and uses magnetic beads to carry DNA to travel through modules to accomplish the multi-step process. The operation can be fully automated, saving time and repetitive hands-on operations and reducing human errors. Using the enclosed reaction system allows parallel processing of different samples and avoids cross-contamination.

## Claims

### Claim 1

A device for performing automated library preparation for next-generation sequencing, comprising:
a) a housing body;
b) at least one integrated reactor disposed inside the housing body, wherein the integrated reactor comprises more than one module wherein a module comprises at least one chamber for receiving solutions and magnetic beads, wherein adjacent chambers are separated by a chamber wall, wherein there is an opening at the apex of the chamber wall for allowing magnetic beads to pass through, wherein a chamber can be connected to at least one multi-functional helper (MFH) element;
c) a robotic hand for operating the MFH element; and
d) a magnet for moving the magnetic beads from one chamber to another chamber.

### Claim 2 (depends on 1)

The device of claim 1, wherein the MFH element has fluid exchange with the connected chamber, wherein the MFH element can function as a solution loader, a mixer and/or a thermally controlled reactor.

### Claim 3 (depends on 1)

The device of claim 1, wherein the MFH element is thermally coupled to a temperature controlling element.

### Claim 4 (depends on 2)

The device of claim 2, wherein the MFH element uses a springy mechanism to move a solution to and from the connected chamber, and wherein the robotic hand is used to move solutions in and out of the MFH element by exerting a squeezing force.

### Claim 5 (depends on 2)

The device of claim 2, wherein the MFH element uses a piston to move a solution to and from the connected chamber, wherein the piston is moved by the robotic hand.

### Claim 6 (depends on 5)

The device of claim 5, wherein the piston and inner wall of the MFH element forms at least one enclosed section for receiving a solution.

### Claim 7 (depends on 1)

The device of claim 1, wherein the integrated reactor can be sealed with a cover.

### Claim 8 (depends on 1)

The device of claim 1, wherein the integrated reactor is disposed in a rotatable seat inside the housing body, wherein the integrated reactor can be rotated by the rotatable seat.

### Claim 9 (depends on 1)

The device of claim 1, wherein the integrated reactor comprises at least two modules selected from the following modules in order:
a first module, having one chamber connected to at least an MFH element;
a second module, having a first chamber, a second chamber and a third chamber connected to at least an MFH element;
a third module, having a first chamber, a second chamber and a third chamber connected to at least an MFH element;
a fourth module, having a first chamber, a second chamber and a third chamber connected to at least an MFH element;
a fifth module, having a first chamber, a second chamber, and a third chamber connected to at least one MFH element; and
a sixth module, having a first chamber, a second chamber, and a third chamber.

### Claim 10 (depends on 9)

The device of claim 9, wherein the integrated reactor comprises optionally the first module, the second module, the third module, the fourth module, optionally the fifth module, and the sixth module.

### Claim 11 (depends on 9)

The device of claim 9, wherein the integrated reactor comprises optionally the first module, the third module, the fourth module, optionally the fifth module, and the sixth module.

### Claim 12 (depends on 9)

The device of claim 9, wherein the integrated reactor comprises optionally the first module, the fourth module, optionally the fifth module, and the sixth module, wherein the third chamber of the fourth module is connected to a two-section MFH element or two MFH elements.

### Claim 13 (depends on 9)

The device of claim 9, wherein the integrated reactor comprises the first module, the fifth module and the sixth module.

### Claim 14 (depends on 9)

A method for performing automated library preparation for next-generation sequencing using a device of claim 9, comprising:
a, loading reaction solutions, magnetic beads and a nucleic acid sample into corresponding chambers or MFH elements;
b, sealing the integrated reactor and disposing it into the housing body;
c, allowing the nucleic acid sample to react in a first reaction solution to obtain a modified DNA;
d, allowing the magnetic beads to bind with the modified DNA;
e, sequentially moving the magnetic beads bound with the modified DNA from one chamber to a neighboring chamber and from one module to a neighboring module by use of the magnet, wherein, when the magnetic beads are moved to a chamber with an MFH element, the MFH element is used to add a solution to and/or mix the solution in the connected chamber; and
f, collecting the library preparation in the last chamber of the last module.

### Claim 15 (depends on 14)

The method of claim 14, wherein each module, if present in the device, is loaded as the following:
the chamber of the first module is loaded with magnetic beads in a bead binding buffer, and the MFH element connected to the first module is loaded with a reaction mix and the nucleic acid sample;
the first and the second chamber of the second module are loaded with a washing solution, the third chamber of the second module is loaded with an end repairing reaction mix, and the MFH element connected to third chamber of the second module is loaded with a bead binding buffer;
the first and the second chamber of the third module are loaded a washing solution, the third chamber of the third module is loaded with a dA-tailing reaction mix, and the MFH element connected to the third chamber of the third module is loaded with a bead binding buffer;
the first and the second chamber of the fourth module are loaded with a washing solution, the third chamber of the fourth module is loaded with a ligation reaction mix, and the MFH element connected to the fourth module is loaded with a bead binding buffer;
the first and the second chamber of the fifth module are loaded with a washing solution, the third chamber of the fifth module is loaded with a PCR reaction mix, and an MFH element connected to the fifth module is loaded with a bead binding buffer; and
the first and the second chamber of the sixth module are loaded with a washing solution, and the third chamber of the sixth module is loaded with an elution buffer.

### Claim 16 (depends on 15)

The method of claim 15, wherein the third chamber of the fifth module is connected to a first MFH element that is thermally controlled and left empty, and a second MFH element loaded with a bead binding buffer.

### Claim 17 (depends on 15)

The method of claim 15, wherein the third chamber of the fifth module is connected to a thermally controlled two-section MFH element, wherein the first section of the MFH element is empty and the second section is loaded with a bead binding buffer.

### Claim 18 (depends on 16)

The method of claim 16, wherein the operation in the fifth module comprises the steps of:
a. moving the magnetic beads into the PCR reaction mix in the third chamber of the fifth module;
b. transferring the PCR reaction mix and the magnetic beads to the first MFH element that is thermally controlled;
c. performing a PCR amplification inside the thermally controlled MFH element to obtain a PCR product;
d. transferring the magnetic beads, the PCR product and the PCR reaction mix back to the third chamber of the fifth module;
e. using the second MFH element to add the bead binding buffer and mix the magnetic beads, the PCR reaction mix and the bead binding buffer in the third chamber of the fifth module; and
f. using the magnet to pellet the magnetic beads and move them to the next module.

### Claim 19 (depends on 17)

The method of claim 17, wherein the operation in the fifth module comprises the steps of:
a. moving the magnetic beads into the PCR reaction mix in the third chamber of the fifth module;
b. transferring the PCR reaction mix and the magnetic beads to the first section of the connected MFH element;
c. performing a PCR amplification inside the first section of the MFH element to obtain a PCR product;
d. transferring the PCR product, the PCR reaction mix, and the magnetic beads in the first section of the MFH element and the bead binding buffer in the second section of the MFH element into the third chamber of the fifth module;
e. using the MFH element to mix the solution and the magnetic beads in the third chamber of the fifth module; and
f. using the magnet to pellet the magnetic beads and move them to the next module.

### Claim 20 (depends on 10, 15)

The method of claim 15, wherein a device of claim 10 is used for the library preparation and the first module is not present, wherein the library preparation starts from the third chamber of the second module, and the third chamber of the second module is loaded with the nucleic acid sample and an end repairing reaction mix, and the connected MFH element is loaded with magnetic beads in a bead binding buffer.

### Claim 21 (depends on 11, 15)

The method of claim 15, wherein a device of claim 11 is used for the library preparation and the first module is present, wherein the third chamber of the third module is loaded with an end repairing and dA-tailing reaction mix.

### Claim 22 (depends on 11, 15)

The method of claim 15, wherein a device of claim 11 is used for the library preparation and the first module is not present, wherein the library preparation starts from the third chamber of the third module, wherein the third chamber of the third module is loaded with the nucleic acid sample, and an end repairing and dA-tailing reaction mix, and the connected MFH element is loaded with magnetic beads in a bead binding buffer.

### Claim 23 (depends on 12, 15)

The method of claim 15, wherein a device of claim 12 is used for the library preparation,
wherein, when the first module is present, the third chamber of the fourth module is loaded with an end repairing and dA-tailing reaction mix;
wherein, when the first module is not present, the library preparation starts from the third chamber of the fourth module, and the third chamber of the fourth module is loaded with a nucleic acid sample, and an end repairing and dA-tailing reaction mix;
wherein a ligation mix and magnetic beads in a bead binding buffer are separately loaded in the connected MFH element or MFH elements.

### Claim 24 (depends on 23)

The method of claim 23, wherein the third chamber of the fourth module is connected to two MFH elements, wherein a first MFH element is loaded with the ligation mix and a second MFH element is loaded with magnetic beads in a bead binding buffer, and wherein the operation in the fourth module comprises the steps of:
a, incubating the nucleic acid sample or nucleic acid-bound magnetic beads with the end repairing and the dA-tailing reaction mix in the third chamber of the fourth module to obtain modified nucleic acids;
b, transferring the ligation reaction mix in the first MFH element to the third chamber of the fourth module to react with the modified nucleic acids;
c, using the second MFH element to add the bead binding buffer and mix the magnetic beads and the solution in the third chamber of the fourth module; and
d, using a magnet to pellet the magnetic beads and move them to the next module.

### Claim 25 (depends on 23)

The method of claim 23, wherein the third chamber of the fourth module is connected to an MFH element with two sections, wherein a first section of the MFH element is loaded with the ligation mix and a second section of the MFH element is loaded with the magnetic beads in a bead binding buffer, and wherein the operation in the fourth module comprises the steps of:
a, incubating the nucleic acid sample or nucleic acid-bound magnetic beads with the end repairing and the dA-tailing reaction mix in the third chamber of the fourth module to obtain modified nucleic acids;
b, transferring the ligation reaction mix in the first section of the connected MFH element into the third chamber of the fourth module to react with the modified nucleic acids;
c, transferring the bead binding buffer in the second section of the connected MFH element into the third chamber of the fourth module;
d, using the connected MFH element to mix the solution and the magnetic beads in the third chamber of the fourth module; and
e, using a magnet to pellet the magnetic beads and move them to the next module.